Daptomycin analogues and a method for the preparation of daptomycin or a daptomycin analogue

ABSTRACT

A method for the synthesis of daptomycin or a daptomycin analog is carried out on a resin to form a linear precursor followed by a serine ligation macrocyclization in solution. Daptomycin analogs can differ from daptomycin by substitution of amino acids residues and/or deletion or addition of amino acid residues. Daptomycin analogs can include a different fatty acid in the side arm of the daptomycin analog.

BACKGROUND OF INVENTION

The emergence of multi-drug resistant bacterial pathogens has created anurgent need for development of effective antibiotics that display newmodes of action against resistant strains. Daptomycin is alipodepsipeptide that is isolated from Streptomyces roseoporus obtainedfrom soil samples from Mount Ararat (Turkey). Daptomycin is the firstnatural product antibiotic launched in a generation and is FDA approvedfor the treatment of skin infections caused by Gram-positive pathogens.Daptomycin has potent bactericidal activity with a unique mode of actionagainst antibiotic-resistant Gram-positive pathogens, includingmethicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistantenterococci (VRE) and vancomycin-resistant S. aureus. Daptomycinundergoes a conformational change on binding to calcium ions to enableinsertion into bacterial membranes and induce membrane leakage as theapparent mode of activity. Drug-resistance to daptomycin in pathogenicbacteria has not been observed. Daptomycin, as shown in FIG. 1, is a13-amino acid cyclic lipodepsipeptide of the nonribosomal peptidefamily, as disclosed in Debono et al., J. Antibiotics 1987, 40, 761-777.It consists of a 31-membered ring of 10 amino acids and a linear 3-aminoacid side-chain modified with an n-decanoyl lipid at the N-terminus.There are two unnatural amino acids, kynurenine (Kyn) and3-methyl-glutamic acid (3-mGlu) within the sequence, along with D-Asn,D-Ser, and D-Ala.

Daptomycin's distinct mechanism of action is a new structural motif forthe development of antibiotics. Only a few daptomycin analogues havebeen produced from biosynthesis, and these include mutations at D-Asn,D-Ser, 3-mGlu and Kyn via genetic engineering of the nonribosomalpeptide synthetase (NRPS) in the daptomycin biosynthetic pathway.Chemo-enzymatic synthesis and semi-synthesis has resulted inmodifications to the lipid chain and the δ-amino group of ornithine. Thepresence of two non-proteinogenic amino acids, Kyn and 3-mGlu, in thecyclic peptide backbone, and the macrolactamization of a 31-membereddepsipeptidic ring render daptomycin a challenging target for totalsynthesis.

To this end an efficient synthetic method to daptomycin, that allows theassembly of the peptide sequence with precision and flexibility canallow a wide variation of daptomycin analogues for the establishment ofthe structure-activity relationship of daptomycin and the preparation ofeffective analogues thereof.

BRIEF SUMMARY

Embodiments of the invention are directed to a method for the synthesesof daptomycin molecules and to analogues thereof. Linear peptides aresynthesized by solid phase peptide synthesis (SPPS) in combination withsolution phase synthesis. Macrocyclization involves intramolecularSerine/Threonine ligation (STL) at the serine site of the sequence.Kynurenine residues are formed from by ozonolysis of tryptophan residueswithin an intermediate sequence.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of daptomycin (1).

FIG. 2 show a reaction scheme for the preparation of a resin boundlinear precursor to daptomycin, according to an embodiment of theinvention, where conditions for transformations a through c are: a)Fmoc-SPPS; b) HATU, DIEA, DMF, 45 min; repeat; and c) DTT, DIEA, DMF, 2h.

FIG. 3 show a reaction scheme for the preparation of azide terminal Kyncomprising tetramer 26, according to an embodiment of the invention,where conditions for transformations a through 1 are: a) TFA, 30 min; b)Fmoc-Thr(tBu)-OH, EDCI, HOBt, DIEA, CH₂Cl₂, 8 h; c) 95% TFA, 30 min; d)TBSCl, imidazole, DMF, 12 h; e) DEA, CH₂Cl₂, 2 h, 80%; f)Fmoc-Asp(tBu)-OH, HATU, DIEA, DMF, 8 h; g) DEA, CH2Cl2, 2 h; h)imidazole-1-sulfonyl azide, CuSO₄.5H₂O, NaHCO₃, MeOH, H₂O, 2 h; i) TBAF,AcOH, THF, 4 h; j) Fmoc-Trp(Boc)-OH, PyBOP, DIEA, CH₂Cl₂, 12 h; k)Pd(PPh₃)₄, N-methylaniline, THF, 4 h; l) (i) O₃, −78° C., CH₂Cl₂; (ii)Me₂S, −78° C. to rt, 2 h.

FIG. 4 show a reaction scheme for the ozonolysis of Fmoc-Trp-OH andFmoc-Trp(Boc)-OH with the indole-N protected with Boc, according to anembodiment of the invention, by the steps of: (i) O₃, −78° C., CH₂Cl₂,10 min; and (ii) Me₂S, −78° C. then rt, 2 h.

FIG. 5 show a reaction scheme for the preparation of Fmc-3-mGlu-OH 10,according to an embodiment of the invention, where conditions fortransformations a through f are: a) LiOH, H₂O/THF, 18 h; b) t-Bu-Br,BTEAC, DMAC, K₂CO₃, 55° C., 24 h; c) TBAF, THF, 1 h; d) NaIO₄, RuCl₃,MeCN/CCl₄/H₂O, 1/1/2, 2 h e) 4 M HCl in dioxane, 0° C. to rt, 50 min;and f) Fmoc-OSu, Na₂CO₃, dioxane/H₂O, 18 h.

FIG. 6 shows the removal of the linear precursor 30 from the resin,according to an embodiment of the invention, by treatment withAcOH/TFE/DCM for 1.5 h.

FIG. 7 show a reaction scheme for the macrocyclization of a resin freelinear precursor to daptomycin, according to an embodiment of theinvention, where conditions for transformations a through c are: a) (i)α,α-dimethoxysalicylaldehyde, PyBOP, DIEA, CH₂Cl₂, 2 h; (ii)TFA/H₂O/PhOH, 1 h; b) pyridine acetate, rt, 4 h; and c) TFA/H2O, 10 min.

DETAILED DISCLOSURE

Embodiments of the invention are directed to daptomycin analogues and amethod to prepare daptomycin or daptomycin analogues. In an embodimentof the invention, daptomycin or an analogue thereof is prepared by aseries of reactions on a bound resin to form a linear precursor to the31-membered cyclic peptide of daptomycin, or a 25 to 37-membered cyclicpeptide of a daptomycin analogue, followed by macrocyclization of thelinear precursor after detachment from the resin by a serine ligation.The preparation of the linear precursor involves a standard Fmoc solidphase peptide synthesis (Fmoc-SPPS) that is modified such that atrityl-resin-linked pentapeptide for daptomycin, or a tetrapeptide,pentapeptide or hexapeptide for a daptomycin analogue, is assembled thatundergoes a HATU coupling with a N-terminal azide substitutedtetrapetide comprising the protected unnatural amino acid Kynurenine(Kyn) followed by: additional couplings with two amino acids fordaptomycin or one to three amino acids for a daptomycin analogue understandard SPPS conditions where the last of the amino acids is serine;reduction of the azide; and additional homologation via Fmoc-SPPS toinclude two amino acids and a fatty acid for daptomycin, or one to threeamino acids and a fatty acid for a daptomycin analogue to form a fattyacid terminal side arm of the linear precursor with a terminalN-protected serine. Upon release from the resin, the linear precursor'sC-terminus is converted to a salicylaldehyde ester, or equivalentaldehyde ester, followed by: deprotection of the serine; deprotection ofother protected functionality of the linear precursor; macrocyclizationvia formation of an N,O-acetal; and hydrolysis of the acetal to formdaptomycin or a daptomycin analogue.

According to an embodiment of the invention, daptomycin analogues canhave the structure:

where X is O, NH, or S, R is H, CH₃, C₂H₅ C₃H₇, or C₄H₉, where R′ is aC₆ to C₂₉ saturated or unsaturated hydrocarbon, and when X is O, R isnot CH₃ or R′ is C₉H₁₈. In an embodiment of the invention, any of theamino acid residues other than those from serine and theH₂NCH(CHRXH)CO₂H amino acid, can be deleted or substituted by anotheramino acid to yield analogue 2. The H₂NCH(CHRXH)CO₂H amino acid can bethreonine, as in daptomycin, or it can be serine,R-3-hydroxy-S,2-aminopropanoic acid, R-3-hydroxy-S-2-aminobutanoic acid,R-3-hydroxy-S-2-aminopentanoic acid, cysteine, thiothreonine,R-3-thio-S-2-aminobutanoic acid, R-3-thio-S-2-aminopentanoic acid,S,R-2,3-diaminoproprionic acid, S,R-2,3-diaminobutanoic acid, orS,R-2,3-diaminopentanoic acid. In an embodiment of the invention, anadditional amino acid can be included in the macrocycle or in the fattyacid terminal side-arm extending from the macrocycle. In an embodimentof the invention R′C(O)NH is a C₇ to C₂₆ saturated fatty acid amide oran unsaturated fatty acid amide, for example, any amide of enanthicacid, caprylic acid, pelargonic acid, capric acid, undecylic acid,lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmiticacid, margaric acid, stearic acid, nonadecylic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoicacid, erucic acid, or docosahexaenoic acid.

In an exemplary embodiment of the invention, as shown in FIG. 2, apreparative method begins with the formation of a resin bound linearprecursor for the preparation of daptomycin, a trityl-resin-linkedpentapeptide (Fmoc-Orn(Boc)-Asp(tBu)-DAla-Asp(tBu)-Gly) 17, assembledvia standard Fmoc-SPPS methods, as can be appreciated by those ofordinary skill in the art. This pentapeptide, is coupled with an azidetetrapeptide including Kyn, N3-Asp(tBu)-Thr-[O-Kyn(Boc,CHO)-Fmoc]-Gly-OH26, using HATU as the coupling reagent.

Tetrapeptide, 26, is prepared from the Boc protected allyl ester ofglycine 18, as shown in FIG. 3. Acidolysis of the Boc group of 18,followed by coupling with Fmoc-Thr(t-Bu)-OH affords the dipeptideFmoc-Thr(tBu)-Gly-OAll. Removal of the t-Bu group, followed bysilylation of the hydroxyl group yields the dipeptide 19, which, afterremoval of the N-terminal Fmoc group with DEA, is coupled withFmoc-Asp(tBu)-OH to form tripeptide 20. Tripeptide 20 undergoes Fmocremoval to amino tripeptide 21, diazo transfer to form 22, and TBSdeprotection to give tripeptide 23. Esterification of 23 withFmoc-Trp(Boc)-OH using PyBOP gives rise to ester 24 withoutepimerization. Palladium-catalyzed deallylation yields ester 25, whichis subjected to ozonolysis to convert Trp to Kyn to yield 26. Theconversion of the indole of Fmoc-Trp-OH to the phenyformamide structureof Fmoc-Kyn-OH upon ozonolysis has been reported without protecting theindole nitrogen with disappointing conversions of about 20%. ByBoc-protecting the indole N, ozonolysis occurs cleanly, in nearlyquantitative yield, as indicated in FIG. 4.

The resin-linked peptide 27 from 17 and 26, as shown in FIG. 2, issubsequently coupled with Fmoc-3-mGlu(tBu)-OH 10 and Boc-DSer(tBu)-OHunder standard SPPS conditions to produce 28. The preparation ofFmoc-3-mGlu(tBu)-OH 10 from protected cyclic amide 5 is shown in FIG. 5.Ring-opening of 5 with LiOH generated 6, which is subsequent esterifiedat the γ-carboxylic acid via tert-butyl bromide to yield 7. Subsequentremoval of the TBS group with TBAF forms 8, followed by oxidation usingsodium periodate and ruthenium(III) chloride to affordBoc-3-mGlu-(tBu)-OH 9. The Boc group of 9 is selectively removed in thepresence of the t-butyl ester by treatment with 4 M HCl in dioxane.After protection with an Fmoc group, the desired (2S,3R)-methyl glutamicacid building block 10. As indicated in FIG. 2, after reduction of theN-terminal azide group of 28 using dithiothreitol (DTT), further peptidehomologation via Fmoc-SPPS affords the fatty acid terminal side arm ofthe target linear sequence 29 bound to the resin.

The resin bound 29 is released from the trityl resin to yield theside-chain-protected linear peptide acid 30 using AcOH/TFE/CH₂Cl₂, asshown in FIG. 6. FIG. 7 illustrates the conversion of the linear peptideacid 30 to the target exemplary daptomycin 1 in solution. In a firststep the C-terminal acid is converted to a salicylaldehyde ester bydirect coupling between the peptide acid andα,α-dimethoxysalicylaldehyde. After subsequent deprotection of otherprotection groups in the linear peptide, salicylaldehyde ester 31 isobtained. The linear side-chain-deprotected peptide salicylaldehydeester 31 is dissolved in pyridine acetate buffer (mol/mol, 1:1) at aconcentration of 5 mM, which results in macrolactamization ineffectively quantitative conversion to form a macrocyclicN,O-benzylidene acetal 32. Treatment of acetal 32 yields daptomycin 1 inhigh yield and can be isolated by semipreparative reverse-phase HPLC inpure form. Surprisingly, the macrocyclization does not require extremedilutions readily occurring cleanly with 31 concentrations of as littleas 5 to more than 50 mM (8.72 to 87.2 g/L) to yield daptomycin 1 as theonly product at all concentrations.

Other daptomycin analogues 2 can be assembled by this method, accordingto embodiments of the invention. Analogues of resin bound pentapeptideprecursor 17 can be prepared with the substitution of one or more aminoacids, selected from any of the 22 standard α-amino acids or anynon-standard α-amino acids for one or more of the amino acids in 17.Analogues of 17 can be tetrapeptides when an amino acid is removed from17 or hexapeptides when an additional amino acid is included with theamino acids of 17. Analogues of 17 can have formed by substituting aminoacids in addition to removing or adding amino acids.

In an embodiment of the invention, the N-terminal azide substitutedtetrapetide comprising the protected unnatural amino acid Kynurenine(Kyn) 26, can be replaced with a tetrapetide that contains the threonineresidue to yield an analogue of 27. In an embodiment of the invention,the threonine can be replaced with H₂NCH(CHRXH)CO₂H where X is O, NH, orS, R is H, CH₃, C₂H₅ C₃H₇, or C₄H₉. The H₂NCH(CHRXH)CO₂H amino acidsubstituted for threonine can be serine, R-3-hydroxy-S,2-aminopropanoicacid, R-3-hydroxy-S-2-aminobutanoic acid, R-3-hydroxy-S-2-aminopentanoicacid, cysteine, thiothreonine, R-3-thio-S-2-aminobutanoic acid,R-3-thio-S-2-aminopentanoic acid, S,R-2,3-diaminoproprionic acid,S,R-2,3-diaminobutanoic acid, and S,R-2,3-diaminopentanoic acid. Inother embodiments of the invention either or both of the non-Kyn,non-H₂NCH(CHRXH)CO₂H amino acid, and non-serine amino acids can besubstituted by and standard or non-standard α-amino acid.

The analogue of 27 is subsequently coupled with Fmoc-3-mGlu(tBu)-OH 10and Boc-DSer(tBu)-OH under standard SPPS conditions to produce ananalogue of 28. The Fmoc-3-mGlu(tBu)-OH can be omitted, substituted withFmoc-Glu(tBu)-OH, substituted with another Fmoc-amino acid, or anadditional amino acid can included before inclusion of theBoc-DSer(tBu)-OH.

The resin bound linear precursor 29 analogue, is completed by theaddition of dithiothreitol (DTT) for the reduction of the N-terminalazide group of the 28 analogue. After reduction, the 28 analogue ispeptide homologated via Fmoc-SPPS affords with the addition of one tothree Fmoc-amino acids and a fatty acid to form the fatty acid amideterminal side arm of resin bound analogue 29. The fatty acid terminalside arm can be that of daptomycin or can have any amino acidssubstituted for the Trp or Asn or can have either amino acid deleted oran additional amino acid can be included before attachment of theterminal fatty acid. The fatty acid can be enanthic acid, caprylic acid,pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylicacid, myristic acid, pentadecylic acid, palmitic acid, margaric acid,stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid,behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid,cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid,melissic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleicacid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid,α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid,docosahexaenoic acid, or any other C₇ to C₃₀ fatty acid.

Methods and Materials

All commercial materials (from Aldrich, Fluka and GL Biochem) were usedwithout further purification. All solvents were reagent grade or HPLCgrade (from RCI or DUKSAN). Anhydrous tetrahydrofuran (THF) was freshlydistilled from sodium and benzophenone. Dry dichloromethane (CH₂Cl₂) wasdistilled from calcium hydride (CaH₂). Daptomycin was purchased fromXiang Bo Biotechnology Co., Ltd. All separations involved a mobile phaseof 0.05% TFA (v/v) in acetonitrile (solvent A)/0.05% TFA (v/v) in water(Solvent B). HPLC separations were performed with a Waters HPLC systemequipped with a photodiode array detector (Waters 2996) using a Vydac218TPTM C18 column (5 μm, 4.6×250 mm) at a flow rate of 0.6 mL/min foranalytical HPLC, Vydac 218TPTM column (10 μm, 10×250 mm) at a flow rateof 4 mL/min for semi-preparative HPLC and Vydac 218TPTM column (10 μm,22×250 mm) at a flow rate of 10 mL/min for preparative HPLC.Low-resolution mass spectral analyses were performed with a Waters 3100mass spectrometer. ¹H and ¹³C NMR spectra were recorded on Bruker AvanceDRX 300 FT-NMR Spectrometer at 300 Hz for ¹H NMR and 75.47 MHz for ¹³CNMR, Bruker Avance DRX 400 FT-NMR spectrometer at 400 MHz for 1H NMR and100 MHz for 13C NMR or Bruker Avance 600 FT-NMR spectrometer at 600 MHzfor ¹H NMR.

Solid-Phase Peptide Synthesis According to Fmoc-Strategy

Synthesis was performed manually on 2-chlorotrityl chloride Resin (resinloading: 0.4 mmol/g). Peptides were synthesized under standard Fmoc/tBuprotocols. The deblock mixture was a mixture of 20/80 (v/v) ofpiperidine/DMF. The following Fmoc amino acids from GL Biochem wereemployed: Fmoc-Ala-OH, Fmoc-DAla-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,Fmoc-DAsn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Val-OH, Boc-DSer(tBu)-OH.

Upon completion of the synthesis, the peptide resin was subjected to acleavage cocktail. The resin was filtered and the combined filtrateswere blown off under a stream of condensed air. The crude product wastriturated with cold diethyl ether to give a white suspension, which wascentrifuged and the ether subsequently decanted. The remaining solid wasready for HPLC purification.

Compound 5:

Compound 5 was prepared by the method of Milne et al., J. Am. Chem. Soc.2006, 128, 11250-11259.

Compound 6:

Compound 5 (3.1 g, 9.02 mmol) was dissolved in 50 mL THF and 1 M aqueousLiOH solution (27 mL, 3.0 equiv.) was added to the solution. Thereaction mixture was stirred at room temperature for 18 h andconcentrated under vacuo. The solution was adjusted to pH=4 with 10%citric acid and extracted with AcOEt (50 mL×3). The combined organicphase was dried over Na₂SO₄ and concentrated under vacuo to givecompound 6 without further purification (3.7 g, 98%).

Compound 7:

Compound 6 (1.0 g, 2.39 mmol) was dissolved in a solution ofbenzyltrimethylammonium chloride (546 mg, 2.39 mmol) in 50 mLdimethylacetamide. K₂CO₃ (9.9 g, 71 mmol) was added to the solutionfollowed by addition of tert-butyl bromide (327 mg, 2.39 mmol). Thereaction mixture was stirred at 55° C. for 24 h. The reaction mixturewas diluted with AcOEt (100 mL), washed with H₂O (50 mL×3) and washedwith brine (50 mL×1). The organic phase was dried over Na₂SO₄,concentrated under vacuo and purified by flash column chromatography onsilica gel (hexane/AcOEt, 7:1) to give compound 7 (687 mg, 69%). ¹H NMR(400 MHz, CDCl₃) δ 4.64 (1H, d, J=7.8 Hz), 3.65-3.66 (1H, m), 3.56-3.58(2H, m), 2.38 (1H, dd, J=4.3 Hz, 14.6 Hz), 2.22-2.28 (1H, m), 2.04 (1H,dd, J=9.2 Hz, 14.5 Hz), 1.43 (18H, s), 0.93 (3H, d, J=6.8 Hz), 0.88 (9H,s), 0.04 (6H, s); ¹³C NMR (75 MHz, CDCl₃) δ 172.3, 155.8, 80.3, 79.2,63.3, 55.1, 40.3, 31.9, 28.5, 28.2, 25.9, 18.3, 15.3, −5.3, −5.4; EI-MS[M+] 418.3; HRMS (EI+) calcd. for C₁₇H₃₅NO₅Si [M-t-Bu]+ 361.2284; found361.2221.

Compound 8:

Compound 7 (500 mg, 1.20 mmol) was dissolved in 10 mL THF and a 1Msolution of TBAF in THF (6.0 mL, 6.00 mmol) was added to the solution.The reaction mixture was stirred at room temperature for 1 h. Thereaction mixture was diluted with AcOEt (50 mL), washed with 1N HCl (25mL×3) and washed with brine (25 mL×1). The organic phase was dried overNa₂SO₄, concentrated under vacuo, and purified by flash columnchromatography on silica gel (hexane/AcOEt, 4:1) to give compound 8 (305mg, 84%). ¹H NMR (400 MHz, CDCl₃) δ 4.88 (1H, d, J=7.5 Hz), 3.55-3.62(2H, m), 3.50-3.54 (1H, m), 2.71 (1H, S), 2.29-2.40 (1H, m), 2.09-2.23(2H, m), 1.44 (9H, s), 1.43 (9H, s), 0.97 (3H, d, J=6.5 Hz); ¹³C NMR (75MHz, CDCl₃) δ 173.0, 156.6, 81.1, 79.7, 63.8, 56.7, 40.1, 31.3, 28.5,28.2, 16.6; EI-MS [M+] 304.2; HRMS (EI+) calcd. for C₁₄H₂₇NO₄ [M-CH2OH]+273.1940; found 273.1927.

Compound 9:

Compound 8 (300 mg, 1.00 mmol) was dissolved in 10 mL of a mixture ofCH3CN/CCl₄/H₂O (1/1/2, v/v/v). NaIO₄ (642 mg, 3.00 mmol) was dissolvedin the above solution. RuCl₃.xH₂O (5 mg, cat) was added to the reactionmixture. The reaction mixture was stirred at room temperature for 3 h.The reaction mixture was diluted with CH₂Cl₂ (50 mL), washed with H₂O(25 mL×3) and washed with brine (25 mL×1). The organic phase was driedover Na₂SO₄, concentrated under vacuo, and purified by flash columnchromatography on silica gel (hexane/AcOEt, 1:1, with 1% AcOH) to givecompound 9 (260 mg, 82%). ¹H NMR (400 MHz, CD₃OD) δ 6.65 (1H, d, J=9.1Hz), 4.29 (1H, d, J=4.0 Hz), 2.52-2.55 (1H, m), 2.35 (1H, dd, J=3.6 Hz,15.8 Hz), 2.09 (1H, dd, J=7.7 Hz, 15.7 Hz), 1.46 (9H, s), 1.45 (9H, s),0.91 (3H, d, J=6.9 Hz); ¹³C NMR (125 MHz, CD₃OD) δ 172.1, 170.5, 155.3,78.9, 77.6, 55.0, 37.4, 30.7, 25.8, 25.4, 12.3; EI-MS [M+] 318.3; HRMS(EI+) calcd. for C₁₁H₁₉NO₆ [M-t-Bu]+ 261.1212; found 261.1205.

Compound 10:

Compound 9 (250 mg, 0.79 mmol) was dissolved in 10 mL 4N HCl in dioxaneat 0° C. The reaction mixture was stirred at 0° C. for 30 min and atroom temperature for another 20 min. The solvent was removed under astream of condensed air. The HCl salt was dissolved in 20 mL H₂O andNa₂CO₃ (420 mg, 3.96 mmol) was added to the solution. The resultingsolution was added dropwise to a solution of Fmoc-OSu (293 mg, 0.87mmol) in 10 mL dioxane at 0° C. The reaction mixture was stirred at roomtemperature for 18 h. The reaction mixture was concentrated under vacuoand adjusted to pH 2 by 1N HCl. The aqueous solution was extracted withEt₂O (50 mL×3). The combined organic phase was washed with brine (25mL×1). The organic phase was dried over Na₂SO₄, concentrated undervacuo, and purified by flash column chromatography on silica gel(hexane/AcOEt, 2:3, with 1% AcOH) to give compound 10 (187 mg, 54% over2 steps). ¹H NMR (400 MHz, CD₃OD) δ 7.79 (2H, d, J=7.5 Hz), 7.67-7.70(2H, m), 7.29-7.40 (4H, m), 4.32-4.41 (3H, m), 4.24 (1H, t, J=6.8 Hz),2.54-2.61 (1H, m), 2.35 (1H, dd, J=15.8 Hz, 6.8 Hz), 2.10 (1H, dd,J=15.7 Hz, 7.6 Hz) 1.44 (9H, s), 0.94 (3H, d, J=6.8 Hz); ¹³C NMR (100MHz, CD₃OD) δ 174.7, 173.3, 158.9, 145.3, 145.2, 142.6, 128.8, 128.2,126.3, 120.1, 81.8, 68.0, 58.5, 40.3, 33.6, 28.3, 15.2; HRMS (ESI+)calcd. for C₂₅H₃₀NO₆ [M+] 440.1995; found 440.2068.

Kyn-Containing Fragment

Compound 18:

Boc-Gly-OH (5.0 g, 28.56 mmol, 1 equiv.) was dissolved in 20 mLanhydrous CH₂Cl₂ to which 2-propenol (33.6 g, 56.90 mmol, 20 equiv.),EDCI (9.8 g, 51.04 mmol, 1.8 equiv.), and DMAP (350 mg, 2.86 mmol, 0.1equiv.) were added. The reaction mixture was stirred at room temperaturefor 6 h. The reaction mixture was diluted with CH₂Cl₂ (250 mL), washedwith 1N HCl (100 mL×3), and washed with brine (100 mL×1). The organicphase was dried over Na₂SO₄, concentrated under vacuo, and purified byflash column chromatography on silica gel (hexane/AcOEt, 4:1) to givecompound 18 (6.0 g, 98%). ¹H NMR (300 MHz, CDCl₃) δ 5.85-5.98 (1H, m),5.25-5.37 (2H, m), 5.00 (1H, m), 4.65 (2H, d, J=5.8 Hz), 3.94 (2H, d,J=5.5 Hz), 1.46 (9H, s)¹³C NMR (75 MHz, CDCl₃) δ 170.2, 155.8, 131.7,118.9, 80.1, 65.9, 42.5, 28.4; HRMS (ESI⁺) calcd. for C₁₀H₁₈NO₄ [M⁺]216.1158; found 216.1230.

Compound 19:

Compound 18 (6.0 g, 27.90 mmol, 1 equiv.) was dissolved in 50 mL TFA.The resulting solution was stirred at room temperature for 30 min toremove the Boc group. TFA was removed under a stream of condensed airleaving a residue of the TFA salt. Fmoc-Thr(tBu)-OH (19.9 g, 50.17 mmol,1.8 equiv.), EDCI (9.6 g, 50.17 mmol, 1.8 equiv.), HOBt (6.8 g, 50.17mmol, 1.8 equiv.) and DIEA (20 mL, 111.60 mmol, 4 equiv.) were mixed in50 mL anhydrous CH₂Cl₂. The resulting solution was added to the TFAsalt. The reaction mixture was stirred at room temperature for 8 h. Thereaction mixture was diluted with CH₂Cl₂ (250 mL), washed with 1N HCl(100 mL×3) and washed with brine (100 mL×1). The organic phase was driedover Na₂SO₄, concentrated under vacuo, and purified by flash columnchromatography on silica gel (hexane/AcOEt, 4:1) to giveFmoc-Thr(tBu)-Gly-OAll (10.2 g, 74%).

Fmoc-Thr(tBu)-Gly-OAll (10.0 g, 20.22 mmol, 1 equiv.) was dissolved in50 mL 95% TFA. The resulting solution was stirred at room temperaturefor 30 min to remove the t-Bu group. TFA was removed under a stream ofcondensed air and the crude compound was dissolved in 50 mL anhydrousDMF. TBSCl (6.2 g, 40.44 mmol, 2 equiv.) and imidazole (4.1 g, 60.66mmol, 3 equiv.) were added to the DMF solution at 0° C. The reactionmixture was stirred at room temperature for 12 h. The reaction mixturewas diluted with AcOEt (250 mL), washed with 1N HCl (100 mL×3), andwashed with brine (100 mL×1). The organic phase was dried over Na₂SO₄,concentrated under vacuo, and purified by flash column chromatography onsilica gel (hexane/AcOEt, 4:1) to give compound 19 (7.8 g, 70%). ¹H NMR(300 MHz, CDCl₃) δ 7.76 (2H, d, J=7.5 Hz), 7.59-7.61 (2H, m), 7.29-7.42(5H, m), 5.84-5.98 (1H, m), 5.82 (1H, d, J=5.9 Hz), 5.25-5.36 (2H, m),4.66 (2H, d, J=5.6 Hz), 4.38-4.43 (3H, m), 4.12-4.26 (3H, m), 4.02 (1H,dd, J=18.2 Hz, 5.1 Hz), 1.13 (3H, d, J=6.3 Hz), 0.91 (9H, s), 0.14 (6H,s)¹³C NMR (100 MHz, CDCl₃) δ 169.9, 169.1, 156.3, 144.0, 143.8, 141.5,131.6, 127.9, 127.2, 125.3, 120.2, 119.2, 68.2, 67.2, 66.2, 59.5, 47.3,41.5, 25.9, 18.4, −4.6, −4.9; HRMS (ESI) calcd. For C₃₀H₄₁N₂O₆Si [M⁺]553.2656; found 553.2728.

Compound 20:

Compound 19 (4.8 g, 8.69 mmol, 1 equiv.) was dissolved in 100 mL of amixture of CH₂Cl₂/Diethylamine (2/1 v/v) and stirred at room temperaturefor 2 h to remove the Fmoc group. The reaction mixture was concentratedunder vacuo and purified by flash column chromatography on silica gel(CH₂Cl₂/MeOH, 20:1) to give the free amine (2.3 g, 80%).Fmoc-Asp(tBu)-OH (5.7 g, 13.85 mmol, 2.0 equiv.), HATU (4.8 g, 12.62mmol, 1.8 equiv.) and DIEA (4.9 mL, 28.13 mmol, 4 equiv.) were mixed in30 mL DMF. The resulting solution was added to the free amine. Thereaction mixture was stirred at room temperature for 8 h. The reactionmixture was diluted with CH₂Cl₂ (250 mL), washed with 1N HCl (100 mL×3)and washed with brine (100 mL×1). The organic phase was dried overNa₂SO₄, concentrated under vacuo, and purified by flash columnchromatography on silica gel (hexane/AcOEt, 2:1) to give compound 7 (3.9g, 77%). ¹H NMR (300 MHz, CDCl₃) δ 7.76 (2H, d, J=7.4 Hz), 7.58-7.61(2H, m), 7.28-7.40 (6H, m), 5.84-5.98 (2H, m), 5.26-5.34 (2H, m), 4.62(2H, d, J=5.6 Hz), 4.46-4.52 (2H, m), 4.34-4.41 (3H, m), 4.23 (1H, t,J=7.0 Hz), 4.12 (1H, dd, J=14.3 Hz, 7.02 Hz), 4.01 (1H, dd, J=18.1 Hz,5.4 Hz), 2.92 (1H, dd, J=16.7 Hz, 4.6 Hz), 2.75 (1H, dd, J=17.0 Hz, 6.3Hz), 1.45 (9H, s), 1.13 (3H, d, J=6.3 Hz), 0.89 (9H, s), 0.09 (6H, s)¹³CNMR (100 MHz, CDCl₃) δ 171.3, 170.8, 169.9, 169.1, 156.3, 144.0, 143.7,141.4, 131.7, 127.9, 127.2, 125.2, 120.1, 119.0, 82.2, 67.7, 67.5, 66.0,58.6, 51.8, 47.2, 41.4, 37.5, 28.1, 25.9, 19.5, 18.0, −4.7, −5.0; HRMS(ESI⁺) calcd. for C₃₈H₅₄N₃O₉Si [M⁺] 724.3551; found 724.3624.

Compound 21:

Compound 20 (3.9 g, 5.39 mmol, 1 equiv.) was dissolved in 100 mL of amixture of CH₂Cl₂/Diethylamine (2/1 v/v) and stirred at room temperaturefor 2 h to remove the Fmoc group. The reaction mixture was concentratedunder vacuo, and purified by flash column chromatography on silica gel(CH₂Cl₂/MeOH, 20:1) to give compound 21 (2.8 g, quant.).

Compound 22:

Compound 21 (2.8 g, 5.39 mmol, 1 equiv.) was dissolved in 100 mL of amixture of MeOH and H₂O (2:1, v/v). Imidazole-1-sulfonyl azide HCl (2.3g, 11.02 mmol, 2 equiv.), NaHCO₃ (4.5 g, 53.58 mmol, 10 equiv.), andCuSO₄.5H₂O (13 mg, 0.05 mmol, 0.01 equiv.) were added to the solutionand the reaction mixture stirred at room temperature for 12 h. Thereaction mixture was concentrated under vacuo, diluted with AcOEt (250mL), washed with 10% citric acid (100 mL×3), and washed with brine (100mL×1). The organic phase was dried over Na₂SO₄ and concentrated undervacuo to give the azido compound 22, which was used in the next stepwithout further purification.

Compound 23:

The crude azido compound 22 was dissolved in 50 mL of a 1M TBAF solutionin THF and AcOH (1/1, v/v). The reaction mixture was stirred at roomtemperature for 4 h. The reaction mixture was diluted with AcOEt (250mL), washed with H₂O (100 mL×3), and washed with brine (100 mL×1). Theorganic phase was dried over Na₂SO₄, concentrated under vacuo, andpurified by flash column chromatography on silica gel (hexane/AcOEt,1:2) to give compound 23 (1.3 g, 58% over 3 steps). ¹H NMR (400 MHz,CDCl₃) δ 7.38 (1H, d, J=7.7 Hz), 7.18-7.20 (1H, m), 5.86-5.96 (1H, m),5.26-5.37 (2H, m), 4.64 (2H, dt, J=5.9 Hz, 1.2 Hz), 4.35-4.45 (3H, m),4.10 (1H, dd, J=17.9 Hz, 5.9 Hz), 4.01 (1H, dd, J=18.0 Hz, 5.6 Hz), 3.46(1H, d, J=2.7 Hz), 3.07 (1H, dd, J=17.0 Hz, 4.8 Hz), 2.68 (1H, dd,J=17.0 Hz, 8.0 Hz), 1.71 (9H, s), 1.20 (3H, d, J=6.4 Hz)¹³C NMR (100MHz, CDCl₃) δ 171.2, 169.9, 169.8, 169.7, 131.7, 119.5, 82.6, 66.9,66.5, 60.2, 57.6, 41.6, 38.4, 28.3, 18.5; HRMS (ESI⁺) calcd. forC₁₇H₂₈N₅O₇ [M⁺] 414.1910; found 414.1983.

Compound 24:

Compound 23 (1.3 g, 3.15 mmol, 1 equiv.), Fmoc-Trp(Boc)-OH (3.6 g, 6.84mmol, 2.2 equiv.), PyBOP (6.5 g, 12.49 mmol, 4 equiv.), and DIEA (2.2mL, 12.62 mmol, 4 equiv.) were mixed in 30 mL anhydrous CH₂Cl₂. Thereaction mixture was stirred at room temperature for 12 h. The reactionmixture was diluted with CH₂Cl₂ (250 mL), washed with 1N HCl (100 mL×3),and washed with brine (100 mL×1). The organic phase was dried overNa₂SO₄, concentrated under vacuo, and purified by flash columnchromatography on silica gel (hexane/AcOEt, 1:1) to give compound 24(2.5 g, 87%). ¹H NMR (300 MHz, CDCl₃) δ 8.12 (1H, d, J=8.1 Hz),7.73-7.76 (2H, m), 7.49-7.64 (4H, m), 7.25-7.40 (8H, m), 6.75-6.92 (1H,m), 5.76-5.87 (1H, m), 5.66 (1H, d, J=7.5 Hz), 5.38-5.41 (1H, m),5.17-5.28 (2H, m), 4.69-4.64 (1H, m), 4.45-4.56 (2H, m), 4.11-4.43 (4H,m), 3.84-4.03 (2H, m), 3.16-3.31 (2H, m), 3.04 (1H, dd, J=16.8 Hz, 4.5Hz), 2.65 (1H, dd, J=17.0 Hz, 7.9 Hz), 1.64 (9H, s), 1.44 (9H, s), 1.15(3H, d, J=6.5 Hz)¹³C NMR (100 MHz, CDCl₃) δ 170.9, 169.6, 169.3, 168.8,168.2, 156.3, 149.6, 143.8, 143.7, 141.4, 135.5, 131.5, 130.4, 127.9,127.2, 125.2, 124.8, 124.5, 122.9, 120.1, 119.1, 119.0. 115.5, 114.9,84.0, 82.3, 70.3, 67.5, 66.2, 59.9, 55.6, 54.5, 47.2, 41.4, 38.2, 28.3,28.1, 27.6, 15.4; HRMS (ESI⁺) calcd. for C₄₈H₅₆N₇O₁₂ [M⁺] 922.3909;found 922.3981.

Compound 25:

Compound 24 (2.5 g, 2.71 mmol, 1 equiv.) and Pd(PPh₃)₄ (627 mg, 0.54mmol, 0.2 equiv.) were combined in anhydrous THF under argon.N-methylaniline (3 mL, 28.00 mmol, 10 equiv.) was added under argon tothe solution. The reaction mixture was stirred at room temperature underargon for 4 h. The reaction mixture was concentrated under vacuo andpurified by flash column chromatography on silica gel (CH₂Cl₂/MeOH,20:1, with 1% AcOH) to give the free carboxylic acid, compound 25 (2.1g, 85%). ¹H NMR (400 MHz, MeOD) δ 8.07 (1H, d, J=7.9 Hz), 7.72 (2H, d,J=6.5 Hz), 7.60-7.64 (5H, m), 7.48-7.53 (5H, m), 7.11-7.38 (5H, m),5.40-5.42 (1H, m), 4.67 (1H, d, J=4.4 Hz), 4.60-4.64 (1H, m), 4.38-4.41(1H, m), 4.30-4.35 (1H, m), 4.07-4.20 (2H, m), 3.88 (2H, br), 3.26 (1H,dd, J=16.0 Hz, 5.8 Hz), 3.11 (1H, dd, J=14.7 Hz, 8.9 Hz), 2.91 (1H, dd,J=16.7 Hz, 5.3 Hz), 2.67 (1H, dd, J=16.7 Hz, 8.3 Hz), 1.54 (9H, s), 1.50(9H, s), 1.19 (3H, d, J=6.2 Hz)¹³C NMR (100 MHz, MeOD) δ 173.1, 172.4,171.2, 171.0, 170.7, 158.3, 150.9, 145.1, 142.5, 133.7, 133.7, 133.1,133.0, 130.0, 129.9, 128.7, 128.1, 126.3, 126.2, 125.5, 125.2, 123.7,120.9, 120.8, 120.8, 120.1, 116.2, 72.3, 68.1, 60.8, 57.7, 55.6, 42.3,38.4, 28.3, 28.2, 16.9; HRMS (ESI⁺) calcd. for C₄₅H₅₂N₇O₁₂ [M⁺]882.3595; found 882.3668.

Compound 26:

The free carboxylic acid, compound 25, (2.1 g, 2.38 mmol, 1 equiv.) wasdissolved in 10 mL CH₂Cl₂ and cooled to −78° C. The resulting solutionwas treated with O₃ at −78° C. for 5 min. Me₂S (2 mL, 27.23 mmol, 12equiv.) was then added at −78° C. The reaction mixture was allowed towarm to room temperature over 2 h. The reaction mixture was concentratedunder vacuo and purified by flash column chromatography on silica gel(CH₂Cl₂/MeOH, 20:1, with 1% AcOH) to give compound 26 as a pair ofrotamers (2.0 g, 95%). ¹H NMR (400 MHz, MeOD) δ 9.24 (1H, s), 8.00 (1H,d, J=6.9 Hz), 7.76-7.79 (3H, m), 7.61-7.66 (9H, m), 7.51-7.56 (5H, m),7.34-7.38 (3H, m), 7.24-7.34 (4H, m), 5.36-5.40 (1H, m), 4.70-4.74 (1H,m), 4.64-4.67 (1H, m), 4.31-4.40 (3H, m), 4.17-4.22 (1H, m), 3.79-4.00(2H, br), 3.41-3.69 (2h, M), 2.87-2.91 (1H, m), 2.64-2.70 (1H, m),1.18-1.41 (18H, m), 0.87-0.91 (3H, m)¹³C NMR (100 MHz, MeOD) δ 171.4,170.4, 169.8, 169.7, 169.7, 169.6, 169.4, 163.6, 165.9, 151.9, 143.9,143.8, 141.2, 134.5, 132.8, 132.4, 132.3, 131.8, 131.7, 131.7, 131.6,131.6, 130.7, 130.6, 129.6, 129.5, 128.9, 128.6, 128.6, 128.5, 127.4,126.8, 124.9, 119.5, 84.3, 84.2, 81.5, 71.2, 71.1, 66.9, 59.3, 56.3,56.1, 49.9, 41.8, 41.6, 40.9, 39.0, 37.0, 31.3, 27.1, 26.9, 26.7, 22.3,19.4, 15.4, 15.3, 13.0; HRMS (ESI⁺) calcd. for C₄₅H′₅₂N₇O₁₄ [M⁺]914.3494; found 914.3567.

Construction of Daptomycin Resin Bound Precursors

Compound 17:

Resin bound compound 17 was synthesized from 2-chlorotrityl resin (250mg, loading: 0.4 mmol/g) using standard Fmoc-SPPS.

Compound 27:

Compound 26 (229 mg, 0.25 mmol), HATU (48 mg, 0.25 mmol), DIEA (109 μL,0.63 mmol) were mixed in 3 mL anhydrous DMF. The resulting solution wasadded to resin bound 17. The reaction mixture was shaken for 45 min. Thesolution was removed by filtration and the coupling was repeated to giveresin bound 27. The solution was removed by filtration and the resinbound 27 was washed with DMF (5 mL×3).

Compound 28:

Resin bound compound 28 was synthesized using standard Fmoc-SPPS from27.

Compound 29:

A solution of dithiothreitol (2M) and DIEA (1M) in DMF (3 mL) was addedto resin bound 28. The reaction mixture was shaken for 2 h to reduce theazido group. The solution was removed by filtration and the resin waswashed with DMF (5 mL×3), followed by standard Fmoc-SPPS to give resinbound 29.

Release from the Resin and Cyclization to Daptomycin

Compound 30:

Resin bound 29 was treated with a mixture of TFE/CH₂Cl₂/AcOH (1/8/1,v/v/v). The solvent was removed under reduced pressure to release thecrude protected peptide 30 from the resin.

Compound 31:

To 50 mg of the crude protected peptide 30 was added α,α-dimethoxy-salicylaldehyde, PyBOP and DIEA in anhydrous CH₂Cl₂ andtreated with TFA/phenol/H₂O (95/2.5/2.5, v/v/v). Preparative HPLCpurification (20-60% CH₃CN/H₂O over 30 min) followed by concentrationunder vacuo and lyophilization afforded compound 31 as a white powder(5.7 mg, 17%).

Compound 32:

A 0.6 mg portion of compound 31 was dissolved in pyridine/acetic acid(1/1, mole/mole) at a concentration of 5 mM at room temperature. Thereaction mixture was stirred at room temperature for 4 h. Aftercompletion of the reaction, the solvent was removed by lyophilization toafford the N,O-benzylidene actual intermediate 32. A portion of theN,O-benzylidene acetal intermediate 32 was isolated by semi-preparativeHPLC purification and characterized by NMR. The NMR spectrum wasrecorded at Bruker Avance 600 FT-NMR spectrometer (600 MHz) equippedwith a cryoprobe, using water suppression (excitation sculpting). HRMS(ESI+) calcd. for C₇₉H₁₀₆N₁₇O₂₇ [M+] 1724.7444; found 1724.7383.

Compound 1: Daptomycin

Without isolation, the N,O-benzylidene acetal intermediate 32 wastreated with TFA/H₂O/TIPS (94/5/1, v/v/v) for 10 min. The solvent wasremoved under a stream of condensed air. Semi-preparative HPLCpurification (20-60% CH₃CN/H₂O over 30 min) followed by concentrationunder vacuo and lyophilization afforded compound 1 as a white powder(0.4 mg, 67%). ¹H NMR was recorded using the same pH condition asdisclosed in Qiu et al., J. Pharm. Sci. 2011, 100, 4225-4233 and Ball etal., Org. Biomol. Chem. 2004, 2, 1872-1878 (5% D₂O in pH 7.8 PBS buffer,0.5 mM). The spectrum was recorded at Bruker Avance 600 FT-NMRspectrometer (600 MHz) equipped with a cryoprobe, using watersuppression (excitation sculpting). The ¹H NMR spectrum obtained was inaccordance with the literatures reported and the authentic sample ofdaptomycin. HRMS (ESI+) calcd. for C₇₂H₁₀₂N₁₇O₂₆ [M+] 1620.7104; found1620.7176.

All publications referred to or cited herein are incorporated byreference in their entirety, including all figures and tables, to theextent they are not inconsistent with the explicit teachings of thisspecification. It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application.

We claim:
 1. A daptomycin analogue, wherein said daptomycin analogue hasthe structure:

wherein X is O, R is H, and R′C(O)— is a residue of palmitic acid,margaric acid, stearic acid, nonadecylic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoicacid, erucic acid, or docosahexaenoic acid.
 2. A daptomycin analogue,wherein said daptomycin analogue has the structure:

wherein where X is O, and R is H, where R′ is a C₉ saturated hydrocarbonand wherein said daptomycin analogue has one to four of the amino acidresidues other than the H₂NCH(CHRXH)CO₂H amino acid, serine, andkynurenine substituted by any natural or unnatural α-amino acid residue,optionally, has two to four additional amino acid residues in thepeptide sequence or macrocyclic ring, or has two to four of the aminoacid residues other than the H₂NCH(CHRXH)CO₂H amino acid, serine, andkynurenine removed, and wherein the macrocyclic ring is a 25 to 37membered ring.
 3. A daptomycin analogue according to claim 2, whereinsaid daptomycin analogue has two to four additional amino acid residuesin the peptide sequence or macrocyclic ring.
 4. A daptomycin analogueaccording to claim 2, wherein said daptomycin analogue has other thanthe H₂NCH(CHRXH)CO₂H amino acid, serine, and kynurenine two to four ofthe amino acid residues removed.